Flowers having new traits are always valued in the flower industry. In particular, the development of plants in which “color”, which is considered to be the most important trait of a flower, has been altered is considered to be industrially important, and various flower colors have been developed through selective breeding using the classical method of crossbreeding. Although crossbreeding is an effective method for selective breeding, since there are genetic restrictions unique to plants, this method has the shortcoming of only being able to use genetic resources possessed by related species capable of crossbreeding. For example, despite many years of selective breeding, violet to blue-colored varieties of roses, carnations, chrysanthemums and lilies, vivid red-colored varieties of gentians and irises, and yellow varieties of morning glories have yet to be produced.
Flower color originates in four types of pigments consisting of flavonoids, carotenoids, chlorophyll and betalain. Among these, flavonoids exhibit a diverse range of colors such as yellow, red, violet and blue. The group that exhibits red, violet and blue color is generically referred to as anthocyanins, and the diverse structure of anthocyanins is one of the causes of the diverse range of flower color. Anthocyanins can be broadly divided into three groups according to their aglycon structure in consideration of the biosynthesis pathway thereof. Flowers having a vivid red color in the manner of carnations and geraniums contain a large amount of pelargonidin-based anthocyanins, while flowers having a blue or violet color contain a large amount of delphinidin-based anthocyanins. The reason for the absence of blue or violet varieties of roses, carnations, chrysanthemums and lilies is because these plants do not have the ability to synthesize delphinidin-based anthocyanins.
In addition to the accumulation of delphinidin, any of (i) the modification of anthocyanin by one or a plurality of aromatic acyl groups, (ii) the presence of a co-pigment such as flavone or flavonol together with anthocyanin, (iii) the presence of iron ions or aluminum ions together with anthocyanin, (iv) a rise in the pH of vacuoles where anthocyanin is localized from neutral to weakly alkaline, or (v) the formation of a complex by anthocyanin, co-pigment and metal ions (and this type of anthocyanin is referred to as metalloanthocyanin) is thought to be required in order for flower color to become blue (see Non-Patent Document 1).
Considerable research has been conducted on flavonoid and anthocyanin biosynthesis, and related biosynthetic enzymes and genes encoding those enzymes have been identified (see Non-Patent Document 2 and FIG. 1). For example, the gene of flavonoid 3′,5′-hydroxylase (F3′5′H), which hydroxylates the B ring of flavonoids required for the biosynthesis of delphinidin, is obtained from numerous plants. In addition, transgenic plants that accumulate delphinidin in the petals thereof causing flower color to change to blue have been produced (see Non-Patent Document 4) by introducing these F3′5′H genes into carnations (see Patent Document 1), roses (see Non-Patent Document 3 and Patent Documents 2 and 3) and chrysanthemums (see Patent Document 4). Such carnations and roses are available commercially.
Flavone is a type of organic compound that is a cyclic ketone of a flavane derivative, and in the narrow sense, refers to the compound 2,3-dehydroflavan-4-one represented by the chemical formula C15H10O2 and having a molecular weight of 222.24. In the broad sense, derivatives classified as flavones are generically referred to as “flavones”. Flavone in the broad sense (flavones) refers to a category of flavonoids that are classified as having a flavone structure for the basic skeleton but not having a hydroxyl group at the 3-position. Examples of typical flavones include apigenin (4′,5,7-trihydroxyflavone and luteolin (3′,4′,5,7-tetrahydroxyflavone). In the description of the present application, the term “flavone” refers to flavones in the broad sense, namely derivatives classified as flavones.
Genes of flavone synthases (FNS) required for biosynthesis of flavone are obtained from numerous plants. Flavones are known to have the effect of changing the color of anthocyanins to a deep blue color when present with anthocyanins, and these FNS genes have attracted attention in the modification of flower color. Simultaneous to the accumulation of delphinidin in flower petals, flavones were also accumulated and flower color changed to an even deeper blue color as a result of introducing F3′5′H and FNS gene into roses not having the ability to synthesize flavones (see Patent Document 5). In addition, since flavones also absorb ultraviolet rays in addition to causing flower color to become blue, they protect plants from ultraviolet rays or function as a visual signal to insects in the case of insect-pollinated flowers. In addition, flavones are also involved in the interaction between plants and soil microbes. Moreover, flavones are also used as an ingredient of foods and cosmetics as components beneficial to health. For example, flavones are said to have anticancer activity, and the ingestion of foods containing large amounts of flavones has been demonstrated to treat and prevent cancer.
In addition, genes that modify anthocyanins and flavones have also been obtained from numerous plants. Although these include glycosyltransferases, acyltransferases and methyltransferases, the following provides a description of glycosyltransferases (GT) that catalyze glycosylation reactions. For example, a gene encoding a protein having activity that transfers glucose to the hydroxyl group at the 3-position of anthocyanin has been isolated from plants such as gentian, perilla, petunia, rose and snapdragon (see Non-Patent Documents 4 to 6 and Patent Document 6). A gene encoding a protein having activity that transfers glucose to the hydroxyl group at the 5-position of anthocyanin has been isolated from such plants as perilla, petunia, gentian, verbena or torenia (see Non-Patent Documents 5 to 7 and Patent Document 7). A gene encoding a protein having activity that transfers glucose to the hydroxyl group at the 7-position of a flavone has been isolated from thale cress (see Non-Patent Document 8). A gene encoding a protein having activity that transfers glucose to the hydroxyl group at the 7-position of baicalein has been isolated from Baikal skullcap, and protein expressed by this gene in Escherichia coli has been reported to catalyze a reaction demonstrating activity that transfers glucose to the hydroxyl group at the 7-position of flavonoids (see Non-Patent Document 9). A gene encoding a protein having activity that transfers glucose to the hydroxyl group at the 3′-position of anthocyanin has been isolated from gentian, butterfly pea and cineria (see Patent Document 8). In addition, a gene encoding a protein having activity that successively transfers glucose to hydroxyl groups at two different locations of the A ring and C ring of anthocyanin has been isolated from rose (see Patent Document 9). A gene encoding a protein having activity that successively transfers glucose to hydroxyl groups at two different locations of the B ring of anthocyanin has been isolated from butterfly pea (see Patent Document 10).
Although the aforementioned glycosyltransferases use UDP-glucose as a sugar donor, glycosyltranferases have recently been identified that use acyl-glucose as a sugar donor. A gene encoding a protein having activity that transfers glucose to the hydroxyl group at the 5-position of anthocyanin 3-glucoside has been isolated from carnation, while a gene encoding a protein having activity that transfers glucose to the hydroxyl group at the 7-position has been isolated from delphinium (see Non-Patent Document 10).
In this manner, a large number of proteins exist as glycosyltransferases having activity that transfers glucose to various hydroxyl groups.
However, a large number of glycosyltransferases are thought to remain for which the function thereof has yet to be identified. For example, a gene encoding a protein having activity that transfers a sugar to the 4′-position of a flavonoid, and a gene encoding a protein having activity that successively transfers a sugar to hydroxyl groups at two locations of the A ring and B ring of a flavonoid have yet to be identified. Although a glycosyltransferase gene derived from Livingstone daisy has been reported to demonstrate activity that transfers glucose to one of the hydroxyl groups at the 4′-position or 7-position of a flavonoid in vitro, the inherent activity of this glycosyltransferase in plants transfers glucose to the hydroxyl group at the 5-position of betanidine (see Non-Patent Document 11).
However, metalloanthocyanins represented by the pigments of dayflower, cornflower, salvia and nemophila are composed of six anthocyanin molecules, six flavone molecules and two metal ion atoms, and each component is assembled to form a stable blue pigment (see FIG. 2 and Non-Patent Document 1). For example, the metalloanthocyanin of nemophila is formed from nemophilin (see FIG. 3), malonyl apigenin 4′,7′-diglucoside (see FIG. 4), Mg2+ and Fe3+. Salvia metalloanthocyanin is formed from cyanosalvianin (see FIG. 5), apigenin 4,7′-diglucoside (see FIG. 6) and Mg2+. According to previous research, all blue flowers that form metalloanthocyanins biosynthesize a flavone in which a sugar is added to the hydroxyl groups at both the 4′-position and 7-position, and the sugar added to that flavone has been determined to play an important role in molecular recognition during metalloanthocyanin formation. The sugar coordinated at the 4′-position of a flavone is important in molecular recognition during formation, while the sugar at the 7-position has been indicated to be involved in the stability thereof (see Non-Patent Document 1). Only after these two sugars have been added to a flavone is a metalloanthocyanin formed which results in the expression of an attractive blue color. In addition, the petals of blue Dutch iris contain a flavone in which a sugar has been added to the 4′-position. In addition, since solubility increases and physical properties change as a result of adding two sugars to flavones, their applications to health foods, pharmaceuticals and cosmetic ingredients are expected to expand.